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4.4.6 - Measuring N2 ﬁxation

Rates of N2 ﬁxation can be measured by a number of techniques to address questions of nodule efﬁciency and nitrogen cycling in agricultural and natural plant systems. Nitrogenase is pivotal for initial reduction of N2 but this same enzyme will also reduce acetylene (C2H2) to ethylene (C2H4). Acetylene is an effective competitor with N2 for nitrogenase so the rate of C2H4 synthesis is proportional to nitrogenase activity. Acetylene reduction gives an instantaneous estimate of the N2 ﬁxation rate. Another instantaneous technique requires flushing nodulated roots with an argon : oxygen gas mixture (79:21) to displace all N2. All electron flux through nitrogenase is then diverted to the reduction of protons to H2 rather than N2 to NH4+ (Equation 2). The rate of H2 evolution by roots can thus be used to estimate nitrogenase activity.

Alternative approaches to ‘instantaneous’ estimates of N2 ﬁxation provide an integrated rate of ﬁxation over periods of hours or days. The proportions of inorganic and organic nitrogen compounds in xylem sap are affected by the ratio of inorganic nitrogen taken up to symbiotic N2 ﬁxation; this can be exploited in genera of legumes in which amides and ureides are major products of N2 ﬁxation. Soybean, for example, exports less than 10% of nitrogen to shoots in the form of ureides when supplied nitrate but more than 80% when all nitrogen is biologically ﬁxed. Thus, relative ureide levels in sap give an estimate of N2 ﬁxation.

Many experiments now rely on 15N-based techniques to obtain an integral of ﬁxation over the life of a plant. These techniques rely on a difference in ratio of the stable isotopes of nitrogen (15N and 14N) in soil and atmosphere (Figure 4.50). The soil must be enriched in 15N relative to the atmosphere — either naturally (the process of denitriﬁcation causes a fractionation of the two isotopes, leaving the soil enriched in 15N) or by artiﬁcial 15N addition. The N2-ﬁxing plant of interest is sampled, together with an adjacent non-N2-ﬁxing plant (e.g. grass) whose 15N enrichment represents that of soil nitrogen. 15N enrichment in digested plant material and soil is analysed isotopically in a mass spectrometer and contribution of biological N2 ﬁxation calculated.

Figure 4.50 Basis of the natural abundance method for assessing the contribution of N2 fixation to legume nutrition. This method entails measuring plant 15N/14N ratio by mass spectrometry. Natural differences in 15N/14N ratio between soil and atmospheric nitrogen are exploited. Legumes to the left and right of the figure each have a unique source of nitrogen, while a test plant in the middle relies on both fixed nitrogen and soil inorganic nitrogen. Plants (left) denied a source of inorganic nitrogen (e.g. nitrate) fix atmospheric nitrogen and therefore have low 15N/14N ratios. Plants without nodules (right) take up only soil-derived nitrogen and are enriched with 15N (high 15N/14N ratios). 15N 'signatures' of these two sets of plants can be used to estimate the relative contributions of soil and atmospheric nitrogen as nitrogen sources in the test plant, and therefore to assess the significance of N2 fixation. (Based on Peoples et al. 1989; reproduced with permission of ACIAR)

A typical ‘good’ rate of ﬁxation for a (non-irrigated) ﬁeld of subtropical legumes in northern Australia is c. 60–100 kg N ha–1 year–1. About the same amount of nitrogen is harvested as seed from a crop of cowpea, soybean or chickpea, so growing these legumes does not add net nitrogen to the soil; it does, however, spare nitrogen which would otherwise be removed at harvest. Irrigated legume-based pastures in temperate Australia or New Zealand ﬁx 250–300 kg N ha–1 year–1 and make a substantial contribution to the low energy costs of agriculture in these regions. Selection of appropriate biological N2 ﬁxers could greatly improve N2 ﬁxation in tropical legume crops.